STRUCTURE AND FUNCTION OF HETERO-MULTIMERIC GLUTAMATE RECEPTORS

异源多聚谷氨酸受体的结构和功能

• 批准号: 9026103
• 负责人: Hiroyasu Furukawa
• 金额: $ 8万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-08 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9026103
• 关键词: AMPA Receptors; Agonist; Allosteric Regulation; Alzheimer' s Disease; Architecture; Baculoviruses; Binding; Biochemical; Brain; Calcium; Complex; Crystallization; Crystallography; Cytosol; Detection; Development; Disease; Early Promoters; Electrophysiology (science); Elements; Extracellular Domain; Family; G-Protein-Coupled Receptors; Gated Ion Channel; Glutamate Receptor; Glutamates; Glycine; Goals; Guidelines; Health; Heterogeneity; Ion Channel; Knowledge; Ligand Binding Domain; Ligands; MK801; Magnesium; Mammals; Mediating; Memantine; Membrane; Membrane Proteins; Mental Depression; Methodology; Methods; Molecular; Molecular Conformation; Molecular Structure; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Neurotransmitters; Parkinson Disease; Pattern; Peptide Hydrolases; Permeability; Phosphotransferases; Physiology; Play; Production; Property; Protein Subunits; Proteins; Public Health; RNA Splicing; Recombinants; Regulation; Research; Resolution; Role; Sampling; Schizophrenia; Seizures; Shapes; Stroke; Structure; Structure-Activity Relationship; Synaptic Transmission; Synaptic plasticity; System; Techniques; Therapeutic; Transmembrane Domain; Treatment Efficacy; Variant; base; conformational alteration; design; dimer; inhibitor/antagonist; insight; nervous system disorder; novel; postsynaptic; presynaptic; protein complex; receptor; research study; screening; stem; structural biology; success; voltage

DESCRIPTION (provided by applicant): The overall goal of the research studies proposed here is to obtain high-resolution structures of intact hetero- multimeric N-methyl-D-aspartate receptors (NMDARs). NMDARs belong to the family of ionotropic glutamate receptors, which mediate the majority of excitatory synaptic transmission in mammalian brains. Dysfunctional NMDARs are implicated in various neurological disorders and diseases including schizophrenia, depression, Alzheimer's disease, and Parkinson's disease. A unique aspect of NMDARs is that they are obligatory hetero- tetramers or higher oligomers composed of GluN1 and GluN2 (A-D) or GluN3 (A-B) subunits. Opening of NMDAR ion channels requires binding of glycine to GluN1 and GluN3 and glutamate to GluN2. To date, structural studies of NMDARs have been limited to the hetero-dimeric structures of the GluN1 and GluN2 extracellular domains. Thus, there is no clear knowledge on how subunits and domains are arranged to form hetero-multimeric ion channels and how transmembrane ion channel pores are shaped to confer specific properties of NMDAR ion channels including high calcium conductance and voltage-dependent magnesium block. Despite various technological breakthroughs, success in crystallographic studies on eukaryotic membrane proteins has been limited due to difficulties in expression, purification, and crystallization stemming from sample heterogeneity and instability. Importantly, there has been no crystal structure of eukaryotic hetero- multimeric membrane proteins that are recombinantly produced to date. The fact that numerous ion channels, G protein-coupled receptors, receptor kinases, and intramembrane proteases implicated in neurological diseases exist as hetero-multimers in native states points to the great need for structural studies on hetero- multimeric membrane proteins. To obtain the first crystal structure of hetero-multimeric ion channels and to understand the structure-function relationship of NMDARs, we will conduct research with the following two aims: Aim 1 is to produce intact hetero-multimeric NMDAR proteins using our novel methodology and to biochemical characterize the homogeneously purified proteins; and Aim 2 is to complete structural analysis of intact NMDARs in complex with various ligands reflecting different functional states by applying cutting-edge techniques in membrane protein crystallography and validate structure-based functional hypotheses by biochemical and electrophysiological experiments. Successful completion of the proposed studies is expected to result in the first crystal structure of a hetero-multimeric ion channel and to provide a mechanistic understanding of NMDARs that are critical in brain physiology and development. Importantly, the structural information obtained here will also provide strategies to develop compounds with therapeutic efficacy in neurological disorders and diseases. Furthermore, these studies on NMDARs will establish fundamental guidelines for crystallography on hetero-multimeric membrane proteins.

描述（由申请人提供）：此处提出的研究的总体目标是获得完整的异酸N-甲基-D-天冬氨酸受体（NMDAR）的高分辨率结构。 NMDAR属于离子型谷氨酸受体家族，它介导了哺乳动物大脑中大多数兴奋性突触传播。功能失调的NMDAR与各种神经系统疾病和疾病有关，包括精神分裂症，抑郁症，阿尔茨海默氏病和帕金森氏病。 NMDAR的一个独特方面是，它们是由Glun1和Glun2和Glun2（A-D）或Glun3（A-B）亚基组成的强制性杂型或更高的低聚物。 NMDAR离子通道的打开需要甘氨酸与Glun1和Glun3和谷氨酸与Glun2结合。迄今为止，NMDAR的结构研究仅限于Glun1和Glun2细胞外结构域的杂二聚结构。因此，尚无清楚的了解如何布置亚基和域以形成异晶体离子通道以及跨膜离子通道孔的形状如何赋予NMDAR离子通道的特定特性，包括高钙电导和电压依赖性镁块。尽管有各种技术突破，但由于样品异质性和不稳定性而引起的表达，纯化和结晶的困难，对真核膜蛋白的晶体学研究的成功受到了限制。重要的是，迄今为止重组产生的真核异源膜蛋白的晶体结构尚无晶体结构。随着天然状态中的异质培养剂的存在，许多与神经系统疾病有关的事实是，与神经系统疾病有关，这一事实表明对异源多种膜膜蛋白的结构研究非常需要。为了获得杂多聚体离子通道的第一个晶体结构并了解NMDAR的结构功能关系，我们将使用以下两个目的进行研究：AIM 1是使用我们的新方法生产完整的异晶体NMDAR蛋白，并使用我们的新方法和生物化学表征同质纯化的蛋白质； AIM 2是通过在膜蛋白晶体学中应用最先进的技术来完成与各种配体的完整NMDAR的结构分析，并通过生化和电生理学实验验证基于结构的功能假设。预计所提出的研究的成功完成将导致杂多聚离子通道的第一个晶体结构，并提供对脑生理和发育至关重要的NMDAR的机械理解。重要的是，此处获得的结构信息还将提供策略，以开发具有治疗疗效的化合物在神经系统疾病和疾病中。此外，这些关于NMDAR的研究将建立杂多媒体膜蛋白晶体学的基本准则。